Cyclooxygenases-1 and-2 (COX-1 and COX-2) catalyze the committed step in the conversion of arachidonic acid to prostaglandins and thromboxane. COX-1 is constitutively expressed in many tissues and appears to play a role in homeostatic functions whereas COX-2 is highly regulated in response to a range of agonists and appears to contribute to pathophysiological responses. Selective inhibition of COX-2 has been exploited for the development of antiinflammatory compounds with reduced gastrointestinal toxicities. The major functional differences between COX-1 and COX-2 are believed to derive from their differential expression rather than from distinct biochemical properties. We recently discovered that COX-2 is able to oxygenate a range of neutral derivatives of polyunsaturated fatty acids (i.e., esters and amides). In particular, we found that 2-arachidonylglycerol (2-AG) is as efficient a substrate for COX-2 as arachidonic acid. COX-2 converts 2-AG into the 2-glyceryl derivative of PGH2, which is subsequently transformed into the glyceryl ester derivatives of prostaglandins and thromboxane. Treatment of macrophages with the calcium ionophore, ionomycin, stimulates the release of 2-AG from endogenous stores followed by its conversion in a COX-2 dependent reaction into the glyceryl ester of PGD2, establishing that this metabolic pathway occurs in intact cells. We propose to utilize the resources of the Research Center for Pharmacology and Drug Toxicology to investigate the production and biological activities of this novel family of eicosanoids. We will 1 ) develop methods for the synthesis and analysis of glyceryl-prostaglandins, and evaluate their stability in the presence and absence of intact macrophages; 2) investigate the pathways that lead to agonist-induced release of 2-AG and conversion to glyceryl-prostaglandins emphasizing the range of agonists that stimulate glyceryl-prostaglandin formation; and 3) probe the interaction of glyceryl-prostaglandins with eicosanoid receptors including prostaglandin receptors, nuclear receptors, and cannabinoid receptors. These experiments will help test our hypothesis that synthesis of glyceryl-prostaglandins represents a selective biochemical and biological function of COX-2.